This proposal's long-range objectives are the design, synthesis and testing of mechanism-based inhibitors which will be effective in vitro and in vivo against M. tuberculosis, and causative agent of tuberculosis. The increasing incidence of tuberculosis worldwide, particularly in the United States, and the recent appearance of multi-drug resistant varieties of M. tuberculosis, makes the development of effective, second generation drugs critical. A mechanism-based inhibition approach has been chosen as most appropriate due to the favorable combination of high specificity, low toxicity and biological efficiency exhibited by such inhibitors. As the most attractive target enzyme for these studies, we have selected dihydrodipicolinate reductase (DHPR), due to its ease of assay, its favorable chemistry and its cloning from BCG (bacille Calmette-Guerin). This enzyme is responsible for a critical early step in the lysine biosynthetic pathway, which has as one of its intermediates, diaminopimelate, an essential component of the mycobacterial cell wall. The products of both of both the asd (aspartate semialdehyde dehydrogenase) and ask (aspartokinase) genes are essential for growth of mycobacteria, and the dual biosynthetic importance of this pathway makes the selection of DHPR as a target especially appropriate. The cloned DHPR gene from BCG will be used to clone the corresponding gene from M. tuberculosis, and this gene will be sequenced. Using this sequence, the gene will be overexpressed in E. coli or M. smegmatis. The enzyme will be purified, and its kinetic and chemical mechanism will be determined. The specificity of the enzyme for substrates and competitive inhibitors will be determined. Attempts will be made to crystallize the homogeneous enzyme, and the three- dimensional structure of the enzyme, and its binary and ternary complexes with substrates will be determined. Mechanism-based inhibitors will be synthesized and tested against the purified enzyme to ensure that all kinetic and mechanistic criteria for such inhibitors are met. Those inhibitors meeting these criteria and exhibiting favorable in vitro properties will be tested against cultures of M. tuberculosis, and other pathogenic bacteria. These studies should provide potent new therapeutics, using targets which have not been suggested or explored previously. Additional enzyme targets for this mechanism-based inhibitor approach exist in this same pathway (succinyldiaminopimelate aminotransferase and diaminopimelate decarboxylase), and similar strategies could be applied to these enzymes.
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